Our laboratory investigates molecular mechanisms that control liver proliferation. Liver specific protein, CCAAT/Enhancer Binding Protein alpha (C/EBPalpha), is a strong inhibitor of liver proliferation. In addition to growth arrest, C/EBPalpha supports differentiation-specific functions of the liver. We and others found that C/EBPalpha inhibits cell proliferation through multiple pathways. Two major pathways of C/EBPalpha -dependent growth arrest in the liver, inhibition of cdks and repression of E2F, are mediated via direct protein-protein interactions and are regulated by phosphorylation of C/EBPalpha. We mapped a single amino acid residue (Serl93), which is critical for C/EBPalpha growth arrest: de-phosphorylation of Serl93 or its mutation to Ala abolish the interactions of C/EBP( with cdks and with E2F-Rb complexes and blocks growth arrest. We identified a signal transduction pathway that regulates growth inhibitory activity of C/EBPalpha through de-phosphorylation of Serl93. The activation of PI3K/Akt pathway by insulin leads to de-phosphorylation of C/EBP( at Ser193, blocks growth inhibitory activity of C/EBPalpha and eliminates negative control of proliferation in the liver. Our recent studies show that insulin/PI3K/Akt pathway has several effects on the biological activities of C/EBPalpha. In addition to the block of growth inhibitory activity of C/EBP(, PI3K/Akt-mediated dephosphorylation of C/EBPalpha 1) increases a portion of C/EBPalpha that activates liver specific promoters and 2) creates a new activity of C/EBP( which is to accelerate cell proliferation. Thus, C/EBPalpha may inhibit or promote cell proliferation depending on its phosphorylation status. The major hypotheses of this application are that liver regulates biological functions of C/EBPalpha by phosphorylation and that the regenerating livers support proliferation and maintain differentiation-specific functions by phosphorylation-dependent switch of biological activities of C/EBPalpha. Three Specific Aims are designed to test these hypotheses. Specific Aim I investigates molecular mechanisms by which de-phosphorylated C/EBPalpha accelerates liver proliferation. Specific Aim II examines the hypothesis that PI3K/Akt pathway increases the ability of C/EBP( to activate liver-specific promoters. In Specific Aim III, we will examine the role of phosphorylation-dependent switch of biological functions of C/EBPalpha in vivo. C/EBPalpha-S193A knock-in mice will be generated and proliferative capacities of the liver will be examined in these animals.